Comparative characterization of OncoPro™ and Wnt-Based media reveals distinct phenotypic and pharmacologic states in patient-derived tumor organoids

Background Patient-derived tumor organoids (PDTOs) are strongly influenced by culture medium. We compared OncoPro™ (OP) Tumoroid Culture medium with conventional Wnt/R-spondin medium (Wnt). This recently developed OP medium offers a standardized, serum-free alternative to Wnt-based formulations.

We performed a comparison of OP and Wnt media across 36 PDTO lines from various malignancies. PDTOs were assessed for establishment success, morphology, transcriptomic profiles (bulk RNAseq and fidelity to public single cell (sc) RNAseq databases) and pharmacologic response to a 60-drug panel.

Adaptation to OP succeeded in 83% (15/18), whereas de novo establishment favored Wnt (44.4% vs 22.2%). Transcriptomic profiling revealed retained tumor-identity but with different epithelial states: Wnt upregulated proliferation/stemness-associated genes (e.g. LGR5) and OP enriched adhesion-associated genes and inflammatory/TGF-β programs. In scRNA databases OP signatures preferentially mapped to malignant epithelial compartments in pancreatic cancer, whereas Wnt signatures were linked to non-malignant epithelium. In colon cancer OP signature was correlated with the malignant iCMS3 subtype. Drug screening demonstrated consistent medium-dependent shifts: Wnt-grown organoids were globally more sensitive, particularly to MAPK-axis inhibitors and apoptosis-sensitizers, while OP-grown organoids exhibited relative resistance

Culture medium composition is a key determinant of PDTO phenotype, transcriptome and drug sensitivity. Wnt supports proliferation-associated genes and drug-sensitive states; OP induces adhesion-associated genes and inflammatory-related programs, with broader resistance and high fidelity of the transcriptomic signature in public scRNAseq databases of colon- and pancreatic cancer. Competing Interest Statement The authors have declared no competing interest. Abbreviations - 3D - three-dimensional - 5-FU - 5-Fluorouracil - AC - adenocarcinoma - Ad-DF+++ - Advanced DMEM/F12 supplemented with 1% GlutaMAX, 1% HEPES, 1% penicillin/streptomycin - AOC - area over the curve - BME - basement membrane extract - BSA - bovine serum albumin - CAF - cancer associated fibroblast - CCA - cholangiocarcinoma - CRC - colorectal carcinoma - CSC - cancer stem cell - CUP - carcinoma of unknown primary - DEG - differentially expressed gene - EBUS-FNA - endobronchial ultrasound fine needle aspiration - FDR - false discovery rate; - GEJC - gastro-esophageal junction carcinoma - GSEA - gene set enrichment analysis; - H&E - hematoxylin and eosin; - iCMS - intrinsic consensus molecular subtype - HNSCC - head-and-neck squamous cell carcinoma - LLOD - lower limit of detection; - NOGR - normalized organoid growth rate - NSCLC - non-small cell lung cancer; - OP - OncoPro™ Tumoroid Culture medium - PBS - phosphate-buffered saline - PDAC - pancreatic ductal adenocarcinoma; - PDTO - patient-derived tumor organoid - RNAseq - RNA sequencing; - scRNAseq - single cell RNA sequencing - UZA - University Hospital Antwerp - UMAP - Uniform Manifold Approximation and Projection.